This proposal is a competitive renewal (CA74305) that concerns the application of chemical approaches to the investigation of the mechanisms and effects of phosphorylation of cell signaling proteins. Protein phosphorylation is now well-accepted to be of critical importance in cell growth, differentiation and human diseases. However, a detailed knowledge is lacking of how protein kinases are regulated, how they recognize their substrates, and what the function of their phosphorylation events are. We are developing and applying three approaches to address these issues. In the first approach, we are using the protein semisynthetic method expressed protein ligation (EPL) method, first described by our lab in collaboration with Tom Muir, to site-specifically introduce post-translational modifications and their mimics into phosphoryl transfer enzymes including low molecular weight protein tyrosine phosphatase (LMW-PTP) and casein kinase 2 (CK2). The effects of these modifications on the enzymatic and cellular behaviors of these semisynthetic proteins will be investigated. In the second approach, we are designing and synthesizing bisubstrate analog ATP-peptide conjugates for use in the structural analysis of protein kinase substrate recognition and regulation, including SR Ser/Thr kinases, Her2/neu tyrosine kinase, MAP kinases, Bub1 kinase, and protein kinase A. In some cases, we will use EPL to incorporate these ATP motifs into recombinant proteins to facilitate protein kinase-protein substrate co-crystallization efforts. In the third approach, we are developing a chemical rescue technique that allows for the rapid gain of function of enzymatic activity for a particular tyrosine kinase. We will apply this method to Src family members and other tyrosine kinases in cellular systems to identify novel targets and signaling actions of these important enzymes. The development and application of these chemical methods should ultimately provide a greater understanding of the roles of protein phosphorylation in cellular function in normal physiologic and disease states, including cancer, endocrine disorders, immune system-related diseases, and cardiovascular pathologies.